1. Transposition and transposable elements

2. Transposable elements
“mobile genetic elements”
comprise 45% of human chromosomal DNA “middle repetitive DNA”
contribute to spontaneous mutation, genetic rearrangements, horizontal transfer of genetic material
aid speciation and genomic change (in bacteria transposons are often associated with antibiotic resistance genes)
cells must depress transposition to insure genetic stability

3. Types of transposable elements
DNA vs. RNA
viral vs. nonviral
replicative mechanism vs. excision mechanism

4. transposon

5. insertion mutation
transposon

6. Discovery of transposons
Barbara McClintock 1950’s Ac Ds system in maize influencing kernel color unstable elements changing map position promote chromosomal breaks
Rediscovery of bacterial insertion sequences source of polar mutations discrete change in physical length of DNA inverted repeat ends: form “lollipops” in EM after denaturation/reannealing

7. Composite bacterial transposons
repeated ends, usually inverted, sometimes direct
repeated ends themselves are IS elements and can independently transpose
ends mobilize all intervening DNA
often antibiotic resistance genes (examples Tn3 (ampicillin), Tn5 (kanamycin), Tn10 (tetracycline)
often reside on plasmids

8. Element 1 Element 2 Transposition? wt 1 and 2 ends- neither trp- tnp-
Basic minimal insertion sequence structure
tnp ORF
ends: genetically required, in cis
tnp (transposase): genetically required, trans-acting
Element 1
Element 2
Transposition? wt
1 and 2
ends-
neither
trp-
tnp-
only 2
1 and 2
only 1

9. Structure of Tn3 4957 bp 3 trans-acting genes: transposase “repressor”
38 bp inverted repeat ends
transposase
tnpA
120 bp “IRS” or res internal resolution site
“repressor”
tnpR
Tn3 resolvase
ampcillin-resistance
bla
2 cis-acting sites:

10. tnpR and res mutations cause accumulation of “co-integrate structure”
direct repeat of Tn
cointegrate

11. 2 types of DNA tranposons
excisive mechanism examples: Tn5, Tn10, P elements
replicative mechanism examples: Tn3, bacteriophage Mu

12. Replicative transposons
orignal cut of transposon is only nick and only one strand is initially ligated
element replicates through itself
produces as intermediate a “co-integrate” structure
co-integrate is resolved by resolvase (as TnpR of Tn3) and at specific site (as res of Tn3)

13. Excisive transposons cut-and-paste mechanism
cut themselves out of original site, producing double strand break
cut target site and ligate to element ends, thereby inserting at new site
original site break repaired usually with sister chromosome, restoring transposon at original site sometimes end healed without transposon, can also be associated with deletion at excision site

14. Source of target site duplication “TSD”
GAC
CTG
CTG
GAC
Staggered cleavage of target
CTG
GAC
Ligation of transposon DNA
GAC
CTG
Repair replication generates short direct repeats

15. “degenerate” transposons
many naturally occurring transposable elements have suffered mutation and are no longer active
some of these may have cis-acting end mutations and cannot be mobilized
others may have intact ends but no transposase: these can be mobilized by a element that is tnp+ (“autonomous” element)
Ac Ds system is an example of latter: Ac (activator) can mobilize Ds (dissociator)
MITEs (minature inverted repeat transposable elements) are nonautonomous DNA elements
SINEs are retrotransposon version (LINEs)

16. Comparison of transposition reactions
Direct transesterification reactions DDE motif transposase (integrase)

17. Comparison of tranposase structural organization

18. Mechanism of transposases and retroviral integrases

20. Classification of retroelements
Have obligate RNA intermediate, use reverse transcriptase (RT, RNA-dependent DNA polymerase)
LTR-retroelements: long terminal repeats Ty1/copia, Ty3/gypsy, retroviruses
Non-LTR-retroelements “retroposons” LINES

22. Characteristics of LTR retroelements
Long terminal repeats: required for replication cycle
Genes: gag, pol, (viruses also have env)
Pol is polyprotein which gives rise to RT (reverse transcriptase), IN (integrase) RH (RNase H), PR (protease)
Forms VLPs virus-like particles
Integrase is functionally and structurally similar to transposase of DNA transposons, DDE motif
Integration gives characteristic TSD

23. LTR element replication
tRNA primer
Multiple template
“jumps”

25. Characteristics of non-LTR retroelements
2 ORFs, orf1, orf2
Variable TSD
ORF2 gives rise to EN, endonuclease, (similar to APE) and RT
Uses target primed reverse transcription TPRT
Can transduce 3’ downstream non-element segments

27. Target-primed reverse transcription
Explains:
Insertions are often 5’ truncated
Transduction of 3’ markers

29. Human L1 (LINE-1) retroelement
15% of human DNA
520,000 copies, only 3-5,000 are full-length
Associated with human disease loci
Transpose specifically in germ line